The complete mitochondrial genome of Echinolaelaps fukienensis provide insights into phylogeny and rearrangement in the superfamily Dermanyssoidea

Background Echinolaelaps fukienensis is the dominant mite species parasitic on the body surface of the genus Niviventer. The mitochondrial genome (mitogenome) has its own independent genetic material and genetic system, and is now widely used in population genetics, genealogical biogeography, phylogeny and molecular evolution studies. Species diversity of the superfamily Dermanyssoidea is very rich, but its mitogenomes AT content is high, and it is difficult to amplify the complete mitogenome by routine PCR. To date, we have only obtained the mitogenomes of 6 species, scarcity on sequence data has greatly impeded the studies in the superfamily Dermanyssoidea. Methods Echinolaelaps fukienensis were collected in 2019 from the body surface of Niviventer confucianus (Rodentia, Muridae) in Yunnan Province. The E. fukienensis mitogenome was determined and analyzed for the first time using the Illumina Novoseq 6000 platform. Phylogenetic analyses of the superfamily Dermanyssoidea were conducted based on the entire mitogenome sequences. Results The E. fukienensis mitogenome was 14,402 bp, which is known the smallest genome of the superfamily Dermanyssoidea, encoding a total of 37 genes, including 13 PCGs, 22 tRNAs, 2 rRNAs and 1 control region. Most protein-coding genes use ATN as the start codon and TAN as the stop codon. AT and GC skew of atp8 genes in E. fukienensis were both 0. The average length of 22 tRNA genes of E. fukienensis was 64 bp, and secondary structures of tRNAs showed base mismatches and missing D-arms in many places. Compared with gene arrangement pattern of the hypothetical ancestor of arthropods, the E. fukienensis mitogenome shows a novel arrangement pattern. Phylogenetic tree supported the monophyly of the superfamily Dermanyssoidea. Echinolaelaps fukienensis being the least genetic distant (0.2762) and most closely related to Varroa destructor. Conclusions This study analyzed comprehensive the structure and evolution of the E. fukienensis mitogenome for the first time, enriches molecular data of the genus Echinolaelaps, which will contribute to further understand phylogeny and rearrangement patterns of the superfamily Dermanyssoidea.


Introduction
The superfamily Dermanyssoidea is a complex group with rich species diversity in Gamasida, which prefer to parasitic life and their hosts include Rodentia, Insectivora, Scandentia, Lagomorpha, Chiroptera and small Carnivores [3].The superfamily Dermanyssoidea is closely related to medicine and important vectors for transmission of epidemic hemorrhagic fever [1][2][3].
Echinolaelaps fukienensis belongs to Arthropoda, Arachnida, Acari, Parasitiformes, Gamasida, Dermanyssoidea and is mainly parasitic on Niviventer fulvescens, Niviventer confucianus, Niviventer andersoni and Niviventer excelsior of the genus Niviventer [4].In 1929, Wang first named E. fukienensis and considered that E. fukienensis belonged to the genus Echinolaelaps [5].Some acarologists have suggested that E. fukienensis belonged to the genus Laelaps because of characteristics of the genus Echinolaelaps were not clearly distinguished from Laelaps and renamed Laelaps fukienensis, but until now taxonomic system of E. fukienensis/L.fukienensis has not formed a consensus [3].Because E. fukiensis has a larger body length compared to other mites, the name of E. fukienensis was used for subsequent analysis in this study.
A typical arthropod mitogenomes contains 37 genes, i.e. 22 tRNA genes (tRNAs), 13 protein-coding genes (PCGs), 2 rRNA genes (rRNAs) and 1 control region of variable length (CR) [6].Control region is also called non-coding region (NCR) or AT-rich region.The extremely compact structure of mitogenome (16-19 kb), rapid evolutionary rate, and matrilineal inheritance have made it important molecular marker for studying the origin of species, interspecific and intraspecific phylogenetic relationships in recent years.Current published mitogenomes of Gamasida in NCBI revealed that mitogenomes of Parasitus wandunqingi, Parasitus fimetorum, Microdiplogynium sp, Quadristernoseta cf.longigynium, Quadristernoseta cf.intermedia retained arrangement pattern of the ancestral arthropod gene order, while mitogenomes of other mite species showed varying degrees of rearrangement [7][8][9][10].In addition, mitogenomes of Euseius nicholsi and Metaseiulus occidentalis showed multiple gene duplications [7], which is a relatively rare phenomenon in arthropod mitogenomes.At present, there are few studies on mitogenomes of the superfamily Dermanyssoidea in the world, and there is still a gap in the study of mitogenomes of the genus Echinolaelaps.Morphological features and mitogenome of E. fukienensis were studied to provide novel insights into rearrangement pattern and phylogeny of the superfamily Dermanyssoidea in this study.

Collection and morphological identification of specimens
Echinolaelaps fukienensis (50 individuals) were collected in 2021 from the body surface of N. confucianus (Rodentia, Muridae) in Yunnan Province, China.Alive host (N.confucianus) trapped were placed individually in pre-marked white cotton bags and transferred to laboratory for species identification and parasitological check.Mites on the body surface of each host were collected and preserved in 95% ethanol at −80˚C prior to DNA extraction.Echinolaelaps fukienensis and their host N. confucianus are preserved at Institute of Pathogens and Vectors, Dali University.
Specimens of E. fukienensis were taken from anhydrous ethanol solution and placed in a petri dish.It was then rinsed with distilled water and fixed onto a microscope slide using Hoyer's medium and cover glass.After air-drying, the specimen was photographed for morphological identification under Leica microscope following Deng's descriptions [3].The morphology image of E. fukienensis were edited using Adobe Photoshop.

DNA extraction, mitogenome sequencing and analysis
Total DNA were extracted from 50 individual mite with DNeasy Tissue kit (QIAGEN) following the manufacture's protocol and was assayed for concentration and purity using NanoDrop, and was sent to Shanghai Winnerbio Technology Co., Ltd.(Shanghai, China) for sequencing using Illumina Novoseq 6000 platform.About 1 μg DNA was sheared into 400-500 bp fragments using a Covaris M220 Focused Acoustic Shearer following manufacture's protocol.Illumina sequencing library were prepared from the sheared fragments, followed by paired-end sequencing (2 × 150 bp) on an Illumina NovaSeq 6000 machine.The raw sequencing reads were quality filtered by fastp (version 0.23.0) to obtain 4 Gb of clean data.Illumina clean reads were assembled using Geneious Primer software.The assembly parameters were minimum overlap 50 bp and minimum overlap identity 98%.tRNA genes were identified with tRNAscan-SE and ARWEN [11,12], protein-coding genes and rRNA genes were identified with Geneious Primer software, BLAST and MITOS [13,14].Base composition and codons were analyzed using Geneious Prime and Codon W respectively [15].Nucleotide composition skew was calculated using the follow formula: AT skew = (A-T) / (A + T) and GC skew = (G-C) / (G + C).
To verify accuracy of sequencing results, cox1 short fragment nucleic acid sequences of E. fukienensis known from the NCBI database were traced and verified, we mapped all sequencing reads to partial cox1 sequences for 1000 iterations using "Map to Reference" function in Geneious Prime and get the same mitogenome sequence.

Mitogenome arrangement and phylogeny
To visualize gene order between mitochondrial genes of E. fukienensis and other 6 known species of the superfamily Dermanyssoidea, we made gene arrangement starting with cox1 for mitogenomes of these species for comparative analysis.Genetic distance of the mitogenomes sequences of 7 species in the superfamily Dermanyssoidea was analyzed using MEGA 11.Meanwhile the Bayesian method (BI method) [16] was used to construct phylogenetic tree based on the complete mitogenomes sequences of 7 species in the superfamily Dermanyssoidea.Optimal model was GTR+I+G model by MrMtgui analysis.In Bayesian analysis, four simultaneous Markov chains were run for 1 million generations and trees were sampled every 100 generations.Phylogenetic relationships of 7 species were inferred from evolutionary tree and genetic distance.

Ethics statement
Niviventer confucianus were handled in strict accordance with good animal practice as defined by relevant national and/or local animal welfare bodies, and animal capture protocols and procedures were approved by Animal Ethics Committees at Dali University (approval # MECDU-201806-11).All collection methods were carried out in accordance with the approved guidelines and regulations.

Morphological characterization of E. fukienensis
Morphological features of E. fukienensis were shown in the following Fig 1 .Morphology of E. fukienensis is ovoid, several cover the entire dorsum, plate with 39 pairs of needle-like setae, arranged as in the genus Laelaps, S8 is 0.065 mm, M11 is 0.159 mm, length of sternal shield is greater than width, genito-ventral shield is expanded after basal coxa IV, genito-ventral shield is widest at VI3, VI4 is located at the posterior margin of genito-ventral shield, the anal shield is fan-shaped, with the middle of the anterior margin is slightly concave inward, adanal stea is smaller located on the transverse line of the posterior margin of the anus, postanal stea is coarse.Each leg coxa are with one coarse spiny seta, coxa IV is shorter, dorsal surface of femur of foot I has a pair of long setae [5].

Protein-coding genes and codon usage
Nine protein-coding genes (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6, cob) are on the heavy strand, and the remaining 4 (nad5, nad4, nad4L, nad1) are on the light strand.The total length of 13 PCGs is 10,793 bp, accounting for approximately 74.9% of mitogenome (14,402 bp), AT content of 13 PCGs is 80.7%, and PCGs with the lowest AT content is cox1 (74.2%) and the highest is cob (88%).The longest sequence among 13 protein-coding genes is nad5 (1683 bp) and the shortest is atp8 (108 bp).The start codon of all 13 protein-coding genes starts with ATN, 11 protein-coding genes use TAN as the stop codon, while the other 2 protein-coding genes, cox2 and nad4, use the incomplete codon T as the stop codon.
The relative synonymous codon usage (RSCU) of 13 PCGs was shown in Table 2. Codon W is used to analyze codon usage corresponding to tRNA genes in the E. fukienensis mitogenome, and a total of 3596 codons were analyzed, and Table 2 shows that UUA (421), UUU (409), AUU (396), and AUA (306) were used most frequently, accounting for 42.6% of all codons used.Use of stop codons was relatively normal, and 2 stop codons UAA and UAG played a normal role in termination of mitogenome.In addition, Leucine (Leu), Phenylalanine (Phe), Isoleucine (Ile) and Serine (Ser) as the main amino acids composing proteins also reflect to some extent that codon has an AT base preference.

rRNA and tRNA genes
As most animals mitogenomes, the E. fukienensis mitogenome has 2 rRNA genes.RrnS is on the heavy strand, 701 bp in size, and rrnL is on the light strand, 1086 bp in size (Table 1 and   1).RrnS is between trnP and trnM, rrnL is between trnC and trnV, both rRNAs are close to control region but not connected, and the interval is the distance of 2 tRNAs.Nucleotide composition of rRNA showed a strong AT bias, with 81.9% and 83.8% AT content in rrnS and rrnL, respectively.The E. fukienensis mitogenome has 22 tRNA genes, of which 12 tRNA genes are on the Jstrand and the remaining 10 tRNA genes are on the N-strand, see Table 1 and Fig

Control region and its stem-loop structure
There is 1 control region (>50 bp) in the E. fukienensis mitogenome, located between trnF and trnY, which contain abundant AT content, reaching 84.7%.To better analyze the nucleotide Structure microsatellite-like (AT) n may be synapomorphy of the family Laelapidae.Meanwhile, multiple palindromic sequences were found in control regions.Palindromic sequences refer to sequences read from the 5' end of a sequence or from the 5' end of its complementary strand are the same, and palindromic sequences have mirror symmetry in DNA or RNA.Sequence size is set for online sites that filter palindrome structure.The length of filtered sequence varies in size, but generally palindrome sequence length is not very long.

Mitochondrial gene arrangement patterns
Gene arrangement has been used as a phylogenetic feature for many lineages, including mesostigmatid mites of previous studies.Gene arrangements were mapped based on gene positions in the superfamily Dermanyssoidea mitogenomes to explore gene arrangements variation among them.Gene arrangements of 3  except V. destructor.Comparison with mitogenome gene order of 3 V. destructor (AY163547 from America), V. destructor (NC004454 from France)) and V. destructor (AP019523 from Japan) have the same genome size and the same gene arrangement pattern but gene content have difference, whereas V. destructor (AY163547 from America) has smaller mitogenome size and gene arrangement pattern difference than the other 2 V. destructor.

Phylogenetic analysis
Phylogenetic tree was constructed based on the mitogenome sequences of 7 species in the superfamily Dermanyssoidea, of which V. destructor belongs to the family Varroidae; E. fukienensis, H. linteyini and C. cf.liui belong to the family Laelapidae; D. gallinae belongs to the family Dermanyssidae; P. chloris and T. melloi belong to the family Rhinonyssidae; Carcinoscorpius rotundicauda and Limulus polyphemus were used as outgroups.Phylogenetic analysis supports the monophyly of the superfamily Dermanyssoidea (Fig 6).Mitogenome data of V. destructor included in this study come from 3 countries, V. destructor (NC004454 from France), V. destructor (AY163547 from America), V. destructor (AP019523 from Japan).V. destructor with different geographical regions have differences in their mitogenomes size, base content, gene order and genetic distance (0.0232), but differences are small.Phylogenetic tree used to analyze genetic distance between mitogenomes of the superfamily Dermanyssoidea (see Table 4).Genetic distance between E. fukienensis and V. destructor (Japan) of the family Varroidae was the closest (0.2762), and that between E. fukienensis and C. cf.liu from the family Laelapidae is the farthest (0.4538).

Discussion
The E. fukienensis mitogenome was obtained for the first time in this study with 37 genes of typical arthropod mitogenomes and 14,402 bp in size, which is known the smallest genome of the superfamily Dermanyssoidea.By comparing mitogenomes of H. linteyini and C. cf.liui of the same family, variation in mitogenomes size is mainly reflected in control region, and the length of control region of E. fukienensis is smaller than that of H. linteyini and C. cf.liui.It is reported that in the process of animal evolution, there is a tendency for mitogenomes to decrease in size, and even during the reduction process, gene loss may occur [17,18].Furthermore, genetic studies have shown that smaller mitogenomes are more advantageous than larger ones in the process of transmission to offspring, implying that natural selection tends to select smaller mitogenomes during evolution.Mitochondria play an important role in cellular energy metabolism, and metabolic differences may affect the evolution of mitogenomes, species with higher metabolic rates tend to have smaller mitogenomes [19].Echinolaelaps fukienensis may have a higher metabolic rate, and theoretically, its mitogenome size is more stable.With the passage of time and environment, the changes in size and internal structure of E. fukienensis mitogenome may indicate that harmful mutations have been overcome, causing mitogenome to evolve towards smaller size to reduce their own material consumption [18,19].Normally, rRNAs genes (rrnS and rrnL) of arthropod mitogenomes are on the N-strand and near the largest control region [20], while rrnS of E. fukienensis is on the J-strand.The E. fukienensis mitogenome had rearrangement.Nad5 has the longest length and atp8 has the shortest length among 13 PCGs, which may be synapomorphies of animal mitogenomes.Surprisingly, base composition of atp8 is A = T and C = G, i.e.AT-skew and GC-skew of atp8 are both 0. Similar situation had not been seen in mites.There also been special situations in codons usage of the E. fukienensis mitogenome.In general, all PCGs began with ATN (ATG, ATA and ATT) as the start codon, except cox1 gene with ATC as the start codon, which is rare in mites, however, it also appeared in Neoseiulus womersleyi and Amphitetranychus viennensis mitogenomes [10,21].The stop codon of most protein-coding genes of E. fukienensis mitogenome is TAA, and only the stop codon of nad3 is TAG, while cox2 and nad4 have incomplete codon T as the stop codon, and the occurrence of incomplete stop codons is common in mites [10], such as the occurrence of TAG and ATA in the stop codon of Euseius nicholsi [22], the occurrence of incomplete T and TA in the stop codon of Psoroptes cuniculi [23], and the occurrence of T and TAG in the stop codon of H. linteyini, which is a member from the superfamily Dermanyssoidea, phenomenon of incomplete stop codons in mites is believed to be completed through post-transcriptional polyadenylation to form TAA to exercise the function of termination codons [20,24].Usually stop codon TAA or TAG can always overlap several nucleotides within downstream tRNA.This situation occurred in Megaloptera as a "backup" to prevent translation read through, and also affects some amino acids usage [24], however, part stop codon of 13 protein-coding genes in the E. fukienensis mitogenome did not overlap with tRNA.This may be relatively small size of mites and short generation times, which led to the high evolutionary rate, and different degrees of gene variation occurred at the mitogenome level.
Most tRNAs secondary structures are typical clover-leaf structures, with only trnC and trnS 1 lacking the D-arm, trnS 1 missing the D-arm is a typical feature of metazoan mitogenomes [24], trnC missing the D-arm is relatively rare, among which Centruroides limpidus, Nymphon gracile, and Achelia bituberculata have trnC missing the D-arm [25], but missing the D-arm or T-arm did not affect its function [26], which is a common occurrence in mites [10,[27][28][29].Truncated tRNAs are related to EF-TU proteins in the nematodes mitogenomes [29].EF-TU1 recognizes tRNAs with missing T-arm, while EF-TU2 recognizes tRNAs with missing D-arm.While the presence of EF-TU genes is essential for nematodes, for how does EF-TU affect the secondary structure of tRNAs?One explanation is that in ancestral species a original single EF-TU gene was duplicated and subsequently co-evolved with the respective discrete mitochondrial tRNAs which may have diverged as genome length decreased, with the final result being a truncated tRNA [30,31].How does truncated tRNA function?There is no clear explanation in arthropod, and there are reports that nuclear tRNAs are recruited into mitochondria [32], which makes it unknown whether the pre-existing truncated tRNAs are functional.However, Juhling et al. demonstrated through computational analysis that mitochondrial tRNAs lacking both the D and T arms are functional in nematodes [31,33].The occurrence of truncated tRNAs in arthropod mitochondria may be beneficial to species evolution, and these truncated tRNAs may also be functionally involved in the transmission of genetic information, and their occurrence in E. fukienensis indicates that species are constantly undergoing natural selection.In addition, a total of 15 mismatches occurred in 22 tRNA genes of the E. fukienensis mitogenome, for which base mismatches in tRNA structure are frequent in other mites, such as acrid mite [34], and base mismatches in stem-loop in tRNA secondary structures of both Arachnid and velvet worms [35,36], and G-U mismatches are important for maintaining the secondary structure of tRNAs, perhaps species evolution has tacitly accepted that G-U pairing is a normal occurrence, other base mismatches that appear in tRNA secondary structure may be an adaptation chosen by mites to adapt to natural selection during the evolutionary process, as this type of base mismatch is widespread in Acarina [25,27].It seems that point mutation exist in the genes of these small animals to constantly adapt to natural selection, although to a lesser extent, it may be a better adaptation process for them.This type of base mismatch can be corrected through editing and will not affect the transport of amino acids, which means that the translation process will not be affected by tRNA base mismatches during protein synthesis [27,37].Perhaps for mites in a constantly changing natural environment, synthesizing corresponding functional genes to meet the needs of life activities under the premise of reducing their own material consumption is an ongoing evolution.Anticodon loop has 7 base pairs in tRNA secondary structure, whereas trnA of the E. fukienensis mitogenome had only 6 pairs, while trnS 2 and trnC had 8 pairs, similar situation also occured in trnS 1 and trnY of Psoroptes cuniculi, trnS 1 and trnK of Phytoseiulus persimilis, and trnA and trnV in Paraleius leontonychus [20,23,26], possibly indicating that base pair number of anticodon loop of tRNAs in mites is not very stable and that this occurrence in mites may be the result of their continued evolution.
Normally, TATA sequences of palindromic sequences have different variance but TATA content is constant, and itself is short palindromic structure and consensus core sequence [38].There are multiple TATA sequences in control region of E. fukienensis, indicating a high trend of variation in control region, and there is a mismatched base T in the stem of control region (marked in red in Fig 4).Correlational studies had shown that control region forms even if every 6 base pairs were mismatched.It is possible that certain key proteins or repair factors exist in E. fukienensis to ignore this simple error and synthesize control regions [39], or it may be that the mitogenomes structure of small mites is continued evolution because mismatched base T was also found in control region of M. occidentalis [40].Curiously, palindromic sequences 5'TACAT and 3'ATGTA were not found in control regions of E. fukienensis mitogenome, but this occurred in control regions of Carpoglyphus lactis and Tyrophagus longior [34,41].Usually palindromic sequences TACAT and ATGTA probably function as recognition sites for the arrest of J-stand synthesis in mammals and fish [34], but their unstable presence in small mites may represent the occurrence of higher convergent evolution in mites.Palindromic sequences were found in control regions are not very long and may be recognition sequences of restriction enzyme [42][43][44].It has been previously suggested that sequence flanking stem-loop structure is highly conserved among arthropods, with 5'TATA and 3'GA (A)T motif, which are presumed to play important role in replication and transcription of mitogenome.5'TATA motif was found in control region (marked with red bases), while GA (A)T was not found in control regions of E. fukienensis mitogenome, and these 2 motifs were also not found in Panonychus citri [45,46].Usually, control region of animal mitogenomes have highly variable, and the extension sequence poly-T is relatively conserved, poly-T (>10 bp) is indispensable for replication origin of mitogenome, and no extension sequence poly-T (>10 bp) was found in control region of the E. fukienensis mitogenome.It is possible that other sequences replaced T extension to provide very important information for replication origin of the E. fukienensis mitogenome without the long fragment T extension.
Echinolaelaps fukienensis belongs to the superfamily Dermanyssoidea, and arrangement pattern of mitogenome is still variation with that of other species in Dermanyssoidea.13 protein-coding genes retain the ancestral arthropods gene order, while tRNA and rRNA showed varying degrees of rearrangement (Fig 5).Studying gene rearrangements of the E. fukienensis mitogenome will provide references for evolution of Acari.We show that frequent rearrangements of tRNA genes in gene arrangement pattern map of the superfamily Dermanyssoidea, which is consistent with the previous research result that tRNA genes are more prone to movement than other genes [28,47].There is a theory that frequent gene rearrangements involving tRNA may be ascribed to the smaller size of tRNA and an unknown mechanism that makes tRNA more mobile [28].Although mechanism of gene rearrangement is unclear, gene rearrangements are important for species evolution.Generally speaking, species within the same family have similar or identical gene arrangement orders.It is easy to classify species into different taxa based on gene rearrangements, such as P. chloris and T. melloi in the family Rhinonyssidae [8], Phyllocoptes taishanensis and Epitrimerus sabinae in the family Eriophyoidea [31].We can infer genetic relationship between different taxonomic category by gene arrangement pattern, which indicates that high-level gene rearrangements may be a typical feature for specific taxon, and species is in continuous evolution [10].It is surprising that the V. destructor mitogenome from different countries has variation in size.The sequences of the V. destructor mitogenome from Japan and France had 99.8% similarity, genome size and gene arrangement pattern is the same.While the V. destructor mitogenome from America are 1199 bp smaller than both from Japan and France and have variation in gene arrangements.As for variation in genome size of the same species, is it the result of geographical differences, climate reasons, etc., or were the data sequenced and annotated from America wrong?This needs further study.We suggest collecting the same species from different regions or different hosts for analysis in the future.Genetic distances and phylogenetic trees revealed that H. linteyini and C. cf.liui are members of the superfamily Dermanyssoidea, clustered together as the same family Laelapidae, while H. linteyini and C. cf.liui, which also are members of the superfamily Dermanyssoidea, had genetic distances greater than that of E. fukienensis and the family Rhinonyssidae.Echinolaelaps fukienensis showed a closer relationship with the family Varroidae, which made us to doubt whether E. fukienensis belongs to the family Laelapidae.Since only one species of Echinolaelaps was involved in this study and only a few species of the superfamily Dermanyssoidea were studied in this study, it is not enough to strongly support taxonomic status of E. fukienensis.However, taxonomic status of E. fukienensis or V. destructor should be problematic from results of genetic distance and phylogenetic tree.Therefore, it is hoped that obtain mitogenomes from more species of the superfamily Dermanyssoidea in the future.Such data would help to better understand phylogenetic relationships of the superfamily Dermanyssoidea.

Conclusions
Structure and evolution of the E. fukienensis mitogenome have been analyzed for the first time, and provided insight into evolutionary relationships of different category level of the superfamily Dermanyssoidea.The E. fukienensis mitogenome was known the smallest in the superfamily Dermanyssoidea, with 37 genes typical of metazoan, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region.The heavy strand encodes 22 genes and the light strand encodes 15 genes.Most PCGs have ATN as the start codon and TAN as the stop codon.However, unlike published structure of Gamasida, AT and GC skew values of atp8 gene of E. fukienensis were both 0, it is rare in Gamasida.The average length of 22 tRNA genes of E. fukienensis is 64 bp, which is slightly smaller than the average length of 66 bp in arthropod tRNA genes, and there are base mismatches and missing the D-arms in tRNA secondary structures.Microsatellite like sequences of (AT) n and multiple palindromic sequences were present in control region of the E. fukienensis mitogenome.Phylogenetic tree supports the monophyly of the superfamily Dermanyssoidea.To obtain a more reliable phylogenetic tree, we need to collect and sequence more representative species of the superfamily Dermanyssoidea and further explore evolutionary mechanism of the superfamily Dermanyssoidea.These mitogenomes will provide novel molecular markers for studying the taxonomy and phylogeny of the superfamily Dermanyssoidea in the future.

Fig
Fig 1).RrnS is between trnP and trnM, rrnL is between trnC and trnV, both rRNAs are close to control region but not connected, and the interval is the distance of 2 tRNAs.Nucleotide composition of rRNA showed a strong AT bias, with 81.9% and 83.8% AT content in rrnS and rrnL, respectively.

Fig 2 .
Fig 2. The complete mitogenome of Echinolaelaps fukienensis.Genes on the outside of the circle are coded on the major or J strand, whereas genes on the inside of the circle are on the complement (minor or N) strand.https://doi.org/10.1371/journal.pone.0288991.g002 2. The size of these tRNAs range from 53 bp (trnC) to 68 bp (trnQ and trnN), with an average length of 64 bp, slightly smaller than the size of arthropod tRNA genes (66 bp).Most tRNAs secondary structure have regular clover-leaf secondary structures, except for trnC and trnS 1 lacking the D-arm, seeFig 3.  Predicted tRNAs secondary structure showed base mismatches and use of

Table 1 . Organization of the Echinolaelaps fukienensis mitogenome.
2rnK, trnL2and trnL 1 all showing 2 G-U mismatches.In addition, trnK uses the unconventional anticodon CUU (CUU instead of the standard UUU), which was found for the first time in the superfamily Dermanyssoidea.Predicted tRNA secondary structure had fully paired amino acid acceptor stem, but in anticodon loop, trnS 2 and trnC appear lengthened by 1 bp each, and there are missing bases between the acceptor stem and D-arms in trnC, which may be a unique feature of the E. fukienensis mitogenome.

Table 2 . Codon usage for 13 PCGs of the Echinolaelaps fukienensis mitogenomes.
4)tps://doi.org/10.1371/journal.pone.0288991.t002Fig3.Putative secondary structures of the 22 mitochondrial tRNA genes of Echinolaelaps fukienensis.https://doi.org/10.1371/journal.pone.0288991.g003composition of control region of E. fukienensis, control region of E. fukienensis was visualized and analyzed.There are multiple stem-loop structures by visual analysis of control region structure in E. fukienensis (see Table3 and Fig4).Structure microsatellite-like (AT) n was found in control region.Structure microsatellite-like (AT) n was present in 2 control regions of Hypoaspis linteyini and Coleolaelaps cf.liui, which also belonging to the family Laelapidae.
V. destructor mitogenomes are mapped for comparison in Fig 5.The E. fukienensis mitogenome showed a unique gene arrangement pattern when compared to hypothetical ancestral arthropod mitogenome gene order.Mitogenome gene arrangement patterns have variation in different species of the superfamily Dermanyssoidea.Fig 5 showed that trnI, trnD, trnC, trnV, trnY, trnF, trnP and trnM of E. fukienensis have undergone translocation, and trnS 2 has undergone transposition and inversion.Compared to genes that have undergone translocation and transposition inversion, most genes of E. fukienensis are conserved state.Genes of the other 6 species of Dermanyssoidea are also mostly conserved and shared atp6-cox3-G and nad3-A-R gene clusters, with difference that rrnS gene of E. fukienensis and V. destructor are on the light strand.In terms of the number of mitochon- drial genes, only Dermanyssus gallinae had 36 genes with missing trnQ.Fig 5 showed that H. linteyini and C. cf.liui as the family Laelapidae shared more gene clusters.Ptilonyssus chloris and Tinaminyssus melloi showed the same pattern of mitochondrial gene arrangement but size difference.Other 6 species of Dermanyssoidea shared nad5-H-nad4-nad4L gene clusters